Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Despite therapy with surgery, radiation and in chemotherapy outcomes of these highly toxic treatments are far from optimal. Further there is increasing evidence of long-term morbidity such as neurocognitive deficits and secondary tumors. In particular patients with high Myc expressing medulloblastoma do very poorly. Thus, there is a critical need for more effective therapies to combat this disease, particularly in the high Myc expressing tumors. Using integrated genomic analysis we have recently identified Polo like kinase 1 (PLK1) as a potential therapeutic target in medulloblastoma. PLK1 is critical for regulation of mitosis and importantly, inhibition of PLK1 preferentially kills cancer cells ove normal cells. However, the functional role of PLK1 in medulloblastoma tumorigenesis is not well understood. We have shown inhibition of PLK1 results in suppression of tumor cell growth and increased sensitivity to ionizing radiation in medulloblastoma cells. The objective of this proposal is to provide pre-clinical validation of PLK1 inhibition as a therapeutic approach in medulloblastoma. The central hypothesis is that PLK1 promotes medulloblastoma cell survival by fostering cell adaptation to Myc oncogene induced replicative stress and promoting repair of DNA damage induced by ionizing radiation. The rationale for this proposal is that once PLK1 as a therapeutic target is better understood in medulloblastoma, novel combination therapeutic strategies can be developed. To address the hypothesis the studies in aim one will determine the role of PLK1 in medulloblastoma tumorigenesis by examining the synthetic lethal interaction of Myc expression with PLK1 inhibition and comparing inhibition of PLK1 in high Myc versus low Myc medulloblastoma. Aim two is designed to establish the therapeutic efficacy and tolerability of PLK1 inhibitors BI6727 and ON-013105 in vivo using multiple cell line derived xenografts, patient derived xenografts and a novel genetic murine model of Myc driven medulloblastoma. Aim three will test the working hypothesis that PLK1 promotes DNA damage repair in irradiated medulloblastoma cells by mediating phosphorylation of Rad 51 and enhancing the tumor initiating cell fraction. The proposed studies will define how PLK1 regulates medulloblastoma tumorigenesis and establish PLK1 as a novel therapeutic target in medulloblastoma by providing the scientific rationale and preclinical data required for early phase clinical studies. Completion of these studies is expected impact medulloblastoma therapy by resulting in novel therapeutic strategies incorporating PLK1 inhibition.